Alzheimer's disease (AD) is the most common form of adult-onset dementia. At present, no reliable biochemical test is available for antemortem diagnosis of AD. The disease is usually diagnosed clinically on the basis of exclusion of other forms of dementia. The diagnosis can only be confirmed irrevocally by neuropathologic examination and the demonstration of large amounts of neuritic (senile) plaques and neurofibrillary tangles (NFT) in particular brain regions (McKhann et al, 1984), the latter appearing to correlate better with the severity and the duration of AD.
Neurofibrillary tangles consist of paired helical filaments (PHF). The microtubule-associated protein tau is a major protein component of PHF and NFT (Brion et al., 1985b; Delacourte and Defossez, 1986; Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986; Kondo et al., 1988).
Tau protein exists in different isoforms, of which 4 to 6 are found in adult brain but only 1 isoform is detected in fetal brain. The diversity of the isoforms is generated from a single gene on human chromosome 17 by alternative mRNA splicing (Himmler, 1989; Goedert et al., 1989; Andreadis et al., 1992). The most striking feature of tau protein, as deduced from molecular cloning, is a stretch of 31 or 32 amino acids, occurring in the carboxy-terminal part of the molecule, which can be repeated either 3 or 4 times. Additional diversity is generated through 29 or 58 amino acid-long insertions in the NH.sub.2 -terminal part of tau molecules (Goedert et al., 1989). For simplicity, all numbering in this patent application refers to the human tau variant htau40 containing all exons (441 amino acids long) according to Goedert et al (1989).
In vivo tau promotes microtubule assembly and stability in the axonal compartment of neurons by interactions involving its microtubule binding domain which is localized in the repeat region of tau (255-381) (Lewis et al, 1990).
In normal circumstances adult brain contains 2 a 3 mol phosphate per mole of tau (Selden and Pollard, 1983; Ksiezak-Reding et al, 1992). Phosphorylation of different sites in normal tau as studied in rat and humans is dependent on the developmental state (Lee et al, 1991; Bramblett et al, 1993; Goedert et al, 1993a). Tau variants of 60, 64 and 68 kDa arising as a consequence of phosphorylation have been detected in brain areas showing neurofibrillary tangles (Delacourte et al, 1990; Goedert et al., 1992; Flament et al., 1990b, Greenberg & Davies, 1990). In tau isolated from PHF (PHF-tau), phosphorylation can occur at several positions (Iqbal et al., 1989; Lee et al., 1991; Hasegawa et al., 1992).
Sofar, the detection of PHF-tau in brain extracts, either via antibodies (Mab Alz50: Ghanbari et al., 1990; Mab Ab423: Harrington et al., 1991; Mab AT120: Vandermeeren et al., 1993; Mab AT180; Mab AT270: International application No. PCT/EP 94/04146 filed on Dec. 14, 1994 and Mab AT8: International application published under WO 93/08302), or via the change in molecular weight (Flament et al., 1990, Delacourte et al., 1993), or else by functional assay (Bramblett et al., 1992) has been used to discriminate dementia with altered cytoskeletal properties from normal aged subjects or from patients with other types of dementia. A combination of monoclonal antibodies each-recognizing non-phosphorylated epitopes of tau has been used to detect the presence of tau and PHF-tau in CSF (Van de Voorde et al., 1995). However, this assay lacks the necessary discriminative power to distinguish AD patients from patients suffering from other neurodegenerative disorders.